Vol.

THE JOURNAL
OF BIOLOGICAL
CHEMISTRY
247, No. 1, Issue of January
10, PP. 45-50,
Printed
in U.S.A.

Studies

1972

on the Action

of Tetracycline

and

Puromycin*
(Received for publication,

SHIGEAKI

TANAKA,

KAZUEI

From

the Department
sylvania i91 O/t

of

IGARASHI,

AND

AKIRA

KAJI

Microbiology, School of ikfedicine, University

of Pennsylvania, Philadelphia, Penn-

It was found that tetracycline inhibits not only the acceptor site
binding but also the donor site binding of phenylalanyl-tRNA
in
the presence of high Mg*
(13 ma/r).
The reaction of puromycin with aminoacyl-tRNA
on the ribosome has been used extensively for studying peptide bond formation. However, it has not been clear whether puromycin approaches the donor site from the side of the acceptor site or not.
By use of the complex having N-acetylphenylalanyl-tRNA
both
on the acceptor and the donor site, we concluded that puromycin
approaches the donor site from the side of the acceptor site.

By means of a convenient method for separating

phenylalanyl puromycin
from diphenylalanyl
puromycin,
it was
found that, upon reaction
with puromycin,
phenylalanyl
transfer RNA bound to ribosomes at5 to 6 mu Mg++ yields
phenylalanyl
puromycin
exclusively
while
phenylalanyltRNA bound at 13 mu Mg++ yields diphenylalanyl
puromycin
as well as phenylalanyl
puromycin.
Tetracycline
inhibited
mostly the binding of phenylalanyl-tRNA
to the acceptor site,
but the binding to the donor site may be inhibited
at 13 mu
Mg++.
Puromycin
reaction
of phenylalanyl-tRNA
bound
to the donor site was inhibited
by the presence of N-acetylphenylalanyl-tRNA
at the acceptor site.

MATERIALS

AND

METHODS

Escherickia co& Extract and Other hlaterials-Preparation

of
ribosomes, tRNA from E. coli B, and aminoacyl-tRNA
have been
described in the previous communications
(1). The ribosomes
were washed three times with a buffer containing 0.1 M Tris-HCl
(pH 7.8), 0.01 M magnesium acetate, 0.06 M potassium chloride,
and 0.5 M ammonium chloride, and
0.006 M P-mercaptoethanol,
were free from aminoacyl-tRNA
transfer factor (T factor) and
initiation factors.
Preparation of T factor and G factor was as
described previously (13).
Binding of [4~Phenylalanyl-tRNA
to Ribosomes and Isolation
of Complex of Ribosmes, poZy(U), and [4C]Phenylalanyl-tRNAA typical binding reaction mixture (0.5 ml) for the isolation of the
complex, unless otherwise specified, contained 50 mM Tris-HCl
(pH 7.2), 6 mM magnesium acetate, 40 mM ammonium chloride,
400 pg of poly(U), 5.2 mg of tRNA containing 8.5 X lo5 cpm of
[14C]phenylalanyl-tRNA,
and 5.8 mg of ribosomes.
This binding
condition was called condition A. In some cases, the binding
reaction was performed under identical conditions except that
the Mg+f concentration
was 13 mM. This was called condition
was carried
B. When the enzymatic binding of aminoacyl-tRNA
out, 85 pg of T factor, 0.2 mM GTP, and 2 ml4 dithiothreitol
were
also added to the above binding mixture.
The reaction mixture
was incubated for 20 min at 22. To separate the complex of
ribosomes, poly(U), and [i4C]phenylalanyl-tRNA
from unbound
[%]phenylalanyl-tRNA,
the mixture was placed on 5 ml of a
linear sucrose gradient solution (5 to 20%) containing 20 mM
Tris-HCl
(pH 7.2), 6 mM or 13 mM magnesium acetate, 40 mM
NH&I, and 6 mM P-mercaptoethanol.
The gradients were centrifuged for 65 min at 48,000 rpm in a Beckman-Spinco
rotor (SW
50.1), and the 70 S ribosome portions were collected as the ribosomal complex.
Reaction of Bound [14C]Phenylalanyl-tRNA
with PuromycinThe reaction mixture (0.5 ml) for the puromycin reaction contained 20 mM Tris-HCl
(pH 7.2), 13 mM magnesium acetate, 80
mM NH&l, 0.2 mM GTP, 24 pg of G factor, 1 mM puromycin, and

It has been suggested that there are two ribosomal sites for the
binding of aminoacyl transfer RNA (l-8).
One of the two sites
is sensitive to puromycin and has been called the donor site (site
2) (5, 7). The other site has been called the acceptor site (site 1)
(5, 7). In the presence of low concentrations
of Mg++ (5 to 6
mM), the bound phenylalanyl-tR.NA
reacted with puromycin in
the absence of G factor; in the presence of relatively high Mg++
(13 mM), two ribosomal sites were presumably
occupied by
phenylalanyl-tRNA
(1). These considerations provided a convenient method of assaying the action of antibiotics
such
as erythromycin,
lincomycin, and streptomycin
(9).
In this communication,
we extended our studies on the ribosomal sites with the method which separates phenylalanyl puromycin from diphenylalanyl
puromycin to elucidate the action of
tetracycline and puromycin.
Concerning the mode of action of
tetracycline, it is believed that tetracycline inhibits the binding
of aminoacyl-tRNA
to the acceptor site of the ribosomes (10-12).
However, results of the NH?-terminal
analysis of polyphenylalanine formed from the complex which was prepared in the
presence of tetracycline suggested that the binding of aminoacyltRNA to the donor site may also be influenced (7). The availability of the method to determine phenylalanyl
puromycin as
well as diphenylalanyl
puromycin quantitatively
made it possible
to measure the percentage of inhibition of the binding of phenylalanyl-tRNA
to the acceptor and the donor site, respectively.
* This research was supported by United States Public Health
Service Grant GM-12,053, National Science Foundation
Grant
GB-18355, and Damon Runyon Memorial Fund for Cancer Research, Grant 799-DT.
45

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SUMMARY

June 14, 1971)

46

Action

of Tetracycline

Vol.

247, No. 1

cline hydrochloride was from California Biochemical Co. Specific activity of [14C]phenylalanine was 375 &i per pmole and the
counting efficiency was lo6 cpm per PCi.
RESULTS

Characterization of Complex Formed under Conditions A and BIt has been postulated that in the presence of 5 to 6 mM Mg++
concentration, phenylalanyl-tRNA
binds to the donor site alone,
and in the presence of 13 my Mg+f concentration,
both donor
and a.cceptor sites are occupied.
Our previous studies, on the
mode of action of antibiotics (9), were dependent on this assumption. It was therefore desirable to obtain the evidence which
would indicate that this is indeed the case. From this assumption it follows that we should have mostly phenylalanyl puromytin when the binding was carried out at low Mgf+ (condition A)
while diphenylalanyl
puromycin as well as phenylalanyl puromytin would be formed when the binding was carried out at high
Mg*
(condition B).
Fig. 1A shows that the puromycin derivative formed under
condition A gives one peak corresponding to a phenylalanyl puromycin upon passage through the Sephadex G-15 column.
As
expected from the function of the G factor, the addition of the
factor did not influence the nature or the quantity of the product
as shown in this figure.
On the other hand, when the complex
was made under condition B, the addition of the G factor and
GTP caused a large increase of formation of phenylalanyl puromycin and diphenylalanyl
puromycin as shown in Fig. 1B.
Effect of Tetracycline on Binding of Phenylalanyl-tRNA--It
has
been proposed that tetracycline has an exclusive inhibitory effect
on the binding of aminoacyl-tRNA
to the acceptor site (l&12).
On the other hand, during the studies on the relationship between
the acceptor site and the donor site, it was observed that, under
certain conditions, tetracycline may also inhibit the binding of
phenylalanyl-tRNA
to the donor site (7). It was therefore
desirable to determine exactly the percentage of inhibition of the
binding of aminoacyl-tRNA
to each site.
In the experiment shown in Table I the ribosomal complex was
prepared under various conditions in the presence or absence of
tetracycline.
The puromycin
derivatives
of [lJC]phenylaline
were formed from these complexes and analyzed with Sephadex
G-15 column.
It should be pointed out that the inhibitory effect
of tetracycline
on the binding reaction was much more pronounced when the binding reaction was carried out in the presence of high Mg++ or T factor.
In order to determine which site was more susceptible to
tetracycline,
the formation
of puromycin
derivatives of the
bound phenylalanyl-tRNA
was studied with or without G factor.
AS shown in Table I the addition of G factor stimulated the
formation
of phenylalanyl
puromycin
derivatives
when the
ribosomal complex prepared at high Mgf+ (13 mM) or in the
presence of T factor was used. Table I also shows the results of
the analysis of the puromycin derivatives formed from these
complexes. The relative amounts of phenylalanyl puromycin and
diphenylalanyl
puromycin were calculated.
With these values,
it was possible to estimate the distribution
of phenylalanyltRNA reactive with puromycin
between the donor and the
acceptor site. The principles used in calculating the distribution
of phenylalanyl-tRNA
between the two sites are (a) phenylalanyl
puromycin formed in the absence of G factor was assumed to be
located at the donor site; (b) diphenylalanyl
puromycin was
derived from phenylalanyl-tRNA
bound to two sites; thus the

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0.3 ml of the fraction containing the ribosomal complex isolated

as described above. The reaction mixture was incubated for 60
min at 22, mixed with 2 ml of 0.01 M Tris-HCl
(pH 7.8), and
shaken well with 6 ml of ethyl acetate. The ethyl acetate extract
(0.5 ml) was mixed with 5 ml of Brays solution (14), and the
radioactivity
was measured by liquid scintillation
counter.
The
ethyl acetate extract (5 ml) wa,s evaporated in a vacuum, redissolved in 0.6 ml of 0.5 M acetic acid, and applied on a Sephadex
G-15 column as described below.
Approximately
70 to 75% of
the bound [14C]phenylalanyl-tRNA
reacted with puromycin under
these conditions.
Separation
of Phenylalanyl
Puromycin
and Diphenylalanyl
Puromycin by Xephadex G-15 Column Chrmatoglaphy-The
puromycin derivatives of phenylalanine
formed as described in the
preceding section were analyzed by Sephadex G-15 column chromatography (15). The puromycin derivative in 0.5 ml of 0.5 M
acetic acid was applied to a Sephadex G-15 column (0.8 x 150 cm)
which had been equilibrated with 0.5 M acetic acid. The elution
was carried out by 0.5 M acetic acid with a flow rate of 1 ml per
12 to 15 min. Each fraction (1 ml) was collected and mixed with
5 ml of Brays solution and the radioactivity
of the [14C]phenylThe elution profile
alanyl puromycin derivatives was measured.
is shown in Fig. 1.
IdentQication of Purcnnycin Derivatives of Phenylalanine-To
identify the nature of two peaks of [%]phenylalanyl
puromycin
derivatives (Fig. l), NHz-terminal
analysis of each peak was performed. Upon dinitrophenylation,
[14C]phenylalanyl puromycin
would give dinitrophenyl
[14C]phenylalanyl
puromycin
which
would be hydrolyzed by 6 N HCl into dinitrophenyl
[14C]phenylalanine and puromycin.
The dinitrophenyl
phenylalanine
is
soluble in ether. Thus, under these procedures, lOO% of radioOn the other hand, under
activity should become ether soluble.
identical conditions [14C]diphenylalanyl
puromycin would yield
only 50% of its radioactivity
as ether-soluble material because of
insolubility
of phenylalanine
into ether. The materials of the
first peak (Fractions 38 to 42 in Fig. 1, A and B) and the second
peak (Fractions 48 to 58 in Fig. 1B) were separately pooled,
dried in a vacuum, and dissolved in 0.2 ml of 1 $Zotriethylamine.
Dinitrophenylation
was started by addition of 0.2 ml of 5%
solution of dinitrofluorobenzene
in alcohol and carried out for 4
hours at room temperature.
After the dinitrophenylation
was
completed, 6 N HCl (0.2 ml) was added to the reaction mixture,
and free dinitrophenol
was removed by ether extraction.
Under
these acidic conditions, most of the dinitrophenyl
[14C]phenylalany1 puromycin derivatives remained in the aqueous layer. The
concentration of HCl of these aqueous layers was brought to 6 N
by the addition of concentrated
HCl, and acid hydrolysis was
performed at 110 for 6 hours. The resulting hydrolysate was
diluted to 0.6 N HCl with water, and ether-soluble materials extracted three times with 5-ml portions of ether. The radioactivities in the combined ether extracts and aqueous layer were
compared.
With material of the first peak, a major portion
(about 90%) of its radioactivity
was converted to ether-soluble
material indicating
that the first peak represents phenylalanyl
puromycin.
When the second peak was subjected to the identical NH*-terminal
analysis, about 53yo of the radioactivity
was
converted to the ether-soluble material showing that the second
peak represented diphenylalanyl
puromycin.
JJateriaZs-Preparation
of N-acetylphenylalanyl-tRNA
was
performed as described previously (16). Puromycin hydrochloride was purchased from Nutritional
Biochemicals, and tetracy-

and Puromycin

Issue of January

X. Tanaka, K. Igarashi,

20

30

40

Fraction

50

60

70

30

20

Number

40

Fraction

50

70
lncubatlon

trne lmin)

of [C]phenylalanyl-tRNA

and enzymatic binding

60

Number

mu Tris-KC1 (pH 7.2), 40 rnM NH&l, 5 mM magnesium acetate,

80 fig of poly(U), 2.5 mg of ribosomes, and 42 fig of tRNA mixture
containing
2 X 10 cpm of N-acetyl[14C]phenylalanyl-tRNA.
After incubation for 15 min at 22, 2.3 mg of tRNA mixture containing 1.2 mpmoles of N-acetyl[laC]phenylalanyl-tRNA
or 3.0
mg of tRNA mixture containing 1.5 voles
of [*C]phenylalanyl
tRNA were added to the above binding reaction mixture and
Mg++ was adjusted to 13 mM. The mixtures were incubated
again for 15 min at 22 to assure the additional binding to the
acceptor site. The ribosomal complex thus prepared was isolated
as described in the text. The reaction mixture for the puromycin
reaction (0.5 ml) of the bound N-acetyl(14C]phenylalanyl-tRNA
was as described in the text and it contained approximately 2,266
cpm of bound N-acetyl[l*C]phenylalanyl-tRNA,
but no G factor.
N-Acetyl[i4C]phenylalanyl-pyromycin
formed is expressed as
percentage of the total bound iV-acetyl[14C]phenylalanyl-tRNA.
O-O,
complex with no additional
[l%]aminoacyl-tRNA
at
the acceptor site; A-A,
complex with N-acetyl[i%]phenylalanyl-tRNA
at the acceptor site; H,
complex with additional [%]phenylalanyl-tRNA
at the acceptor site.

TABLE

of tetracycline on nonenzymatic

47

IO

FIQ. 1 (left and center).

Sephadex G-15 column chromatography
of puromycin derivatives of [~4C]phenylalanine.
The puromycin
derivatives of [C]phenylalanine
prepared under Conditions A
(5,520 cpm) or B (20,292 cpm) were extracted in ethylacetate,
the ethylacetate was evaporated and dissolved in 0.6 ml of 0.5
M acetic acid. The solution was then poured onto a column
(0.8 X 150 cm) of Sephadex G-15 which had been equilibrated
with 0.5 M acetic acid. The sample was eluted with 0.5 M acetic
acid with flow rate of 1 ml every 12 to 15 min. Fractions (1 ml)
were collected and l-ml aliquot was mixed with 5 ml of Brays
solution (14) and the radioactivity
was counted.
O-0,
puromycin derivatives formed in the presence of G factor; O- - -0,
puromycin derivatives formed in the absence of, G factor. A and
B represent puromycin
derivatives formed under Conditions A
and B, respectively.
FIG. 2. (right). Effect of additional binding of aminoacyl-tRNA
on the puromycin reaction of the donor site-bound N-acetyl[iV]phenylalanyl-tRNA.
For the preparation
of the ribosomal
complex having N-acetyl[14C]phenylalanyl-tRNA
mostly at the
donor site, the binding reaction mixture (0.4 ml) contained 40

Effects

and A. Kaji

to ribosomes

and

puramycin

reaction

of bound

[W]phenylalanyl-tRNA

The reaction mixture for the preparation of the ribosomal complex was described in the text except that it contained 4 mg of ribosomes, 32Opg of poly(U), and 1.8 X 106 cpm of [14C]phenylalanyltRNA (4 mg of tRNA); in some cases, 85rg of T factor, 2 mM dithiothreitol, and 0.2 mu GTP were added. The isolation of the
complex of [14C]phenylalanyl-tRNA,
poly(U), and ribosomes, for-

Binding condition of [W]phenylalmyl-tRNA

Bound
- I~W)phtmylalanyl-tRNA

T factor

mation of puromycin derivatives of [%]phenylalanine,

and the
column chromatography
of the puromycin derivatives were carried out as described in the text. Where indicated 15 ag of G
factor was added to the reaction mixture for the formation of puremycin derivatives.

5 x 10-d

G factor

CPm

8,850

7,500

+
-

12,600

+
-

6,190
6,640
6,020
6,020
7,680
11,110
6,100
6,160
5,300
11,400
3,890
4,930

13

5 x 10-d

13

Puromycin derivatives formed

(percentage of total)
Phenylalanyl
puromycin

Diphenylalanyl
puromycin

CM

5 x 10-d

formed

-_

t&w

puromycin

.-

Tetracycline

__

Total phenylalanyl

8,580
17,580

+
-

10,430

+
+

92
89
92
90
64

58
96
94
49
47
94
91

8
11
8
10
36
42
4
6
51
53
6
9

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I
IO

10, 1972

Action

48
II

TABLE

Tetracycline

inhibition

of Tetracycline

of binding

of phenylalanyl-tRNA

Binding

Mg++

T
factor

m.l

6
6
6
6
13
13

Calculated
amount
of
puromycin-reactive
[4C]phenylalanyltRNA
at each site

conditions

Tetracycline

+
+
-

DOnOr
site

Acceptor
site

69

18

97

cpl!

M
-

Acceptor
site

DOnOr
site

Percentage
of
hnhibition
of binding
,f [rC]phenylalanyltRNA
to each site
by tetracycline

5 x 10-4
0
5 x lo-
0
5 x 10-d

GO60
5839
7402
6041
5618
3878

580
181
3709
119
5782
1052

31

82

a Data were derived from Table I.

radioactivity
of this fraction was divided by two and equally
distributed to the acceptor and donor site; and (c) phenylalanyl
puromycin dependent on the presence of G factor was allotted
to the acceptor site. The results of these calculations are summarized in Table II.
It is clear from Table II that the effect of tetracycline
was
mainly on the acceptor site when the binding reaction was carried
out at 6 mM Mg++ or in the presence of T factor.
On the other
hand, when the binding reaction was carried out at 13 mM Mg,
the effect of tetracycline
on the binding of phenylalanyl-tRNA
to the donor site became significant. However, even under these
conditions, the inhibition
of the binding of phenylalanyl-tRNA
to the acceptor site was much larger than that to the donor site.
It should be noted that the assumption (a) used in calculating
these values may not necessarily be correct in view of the fact
that a fair amount of nonenzymatic
translocation
of diphenylalanyl-tRNA
takes place (17). If one assumes that similar
nonenzymatic translocation takes place with phenylalanyl-tRNA

Vol. 247, No.

bound at the acceptor site, the inhibitory

effect of tetracycline
at the donor site becomes less than a few percentage even in the
presence of 13 mM Mg++.
In view of the fact that approximately
equal amounts of phenylalanyl-tRNA
are bound to donor and
acceptor site in the presence of 13 mM Mg++ (2), nonenzymatic
translocation
of phenylalanyl-tRNA
is probably not as much as
that with diphenylala,nyl-tRNA.
This is because non-enzymatic
translocation
of phenylalanyl-tRNA
would give a far greater
amount of phenylalanyl-tRNA
at the acceptor site. At any rate,
one can conclude that a major action of the inhibitory effect of
tetracycline is on the acceptor site. It can also be seen in this
table that the stimulation
of the binding of [r4C]phenylalanyltRNA by T factor was mainly on the acceptor site in confirmation
of our previous conclusion with the experiment
where NH%terminal analysis of polyphenylalanine
was employed to determine the amount of bound phenylalanyl-tRNA
at these sites (2).
Studies on Puromycin
Action and Influence of AminoacyltRNA at Acceptor Site on Reactivity of Aminoacyl-tRNA
Bound
at Donor Site-Since
the puromycin reaction with the donor sitebound aminoacyl-tRNA
is analogous to the peptide bond formation between aminoacyl-tRNAs
bound at the donor and acceptor
sites (B-20),
it is reasonable to assume that puromycin would
attack the aminoacyl-tRNA
at the donor site from the side of
the acceptor site. If this assumption is correct, one would
expect that the presence of nonreactive aminoacyl-tRNA
at the
acceptor site would hinder the reactivity of the aminoacyl-tRNA
at the donor site. The report contrary to this expectation (21)
prompted us to examine this possibility with the present system.
In the experiment described in Table III, the complex of [Clphenylalanyl-tRNA,
poly(U), and ribosomes was prepared at
5 mM Mg++.
This complex was then incubated with or without
N-acetylphenylalanyl-tRNA
or phenylalanyl-tRNA
at 13 mM
Mg++.
Under these conditions,
[W]phenylalanyl-tRNA
or
N-acetyl[W]phenylalanyl-tRNA
and [r4C]phenylala.nyl-tRNA
were at the acceptor and the donor site, respectively
Using
these complexes, the formation of puromycin
derivative was
examined.
It is clear from this table that the amount of puromycin derivative of [14C]phenylalanine formed in the absence of
the G factor was greatly reduced when [W]phenylalanyl-tRNA
or N-acetyl[r2C]phenylalanyl-tRNA
were added to the reaction
mixture for the additional
binding of aminoacyl-tRNA
at the
acceptor site. Since the [Wlphenylalanyl-tRNA
was bound almost exclusively at the donor site, G factor did not appreciably
stimulate the puromycin reaction of this [i4C]phenylalanyl-tRNA
when no additional aminoacyl-tRNA
was bound at the acceptor
site. On the other hand, in the presence of [12C]phenylalanyltRNA at the acceptor site, [W]phenylalanyl-[r2C]phenylalanyltRNA was formed and located at the acceptor site. Consequently, the puromycin reaction of this diphenylalanyl-tRNA
was dependent on G factor.
When N-acetyl[W]phenylalanyltRNA was bound at the acceptor site, no diphenylalanyl-tRNA
could be formed and, consequently, G factor had no appreciable
stimulation
on the formation of puromycin derivative of [Clphenylalanine.
Thus, the inhibitory
effect of N-acetyl[W]phenylalanyl-tRNA
at the acceptor site could be attributed solely
to the steric hindrance.
The data presented in Fig. 2 shows similar effects of the [Clphenylalanyl-tRNA
and N-acetylphenylalanyl-tRNA
present at
the acceptor site on the puromycin reaction of N-acetyl[r4C]phenylalanyl-tRNA
bound at the donor site. Since the peptide
bond formation takes place much more rapidly with N-acetyl-

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The calculations of the amount of the puromycin-reactive

[*Clphenylalanyl-tRNA
at the acceptor and the donor site were carried out as follows.
For example, from the ribosomal complex
formed in the presence of 6 mM Mg*, 6190 cpm of the puromycin
derivatives of [lQZ]phenylalanine
were formed in the absence of
the G factor (line 1 in Table I). Of this, 92yo (5695 cpm) were
phenylalanyl puromycin and 8% (495 cpm) were diphenylalanyl
puromycin.
Since diphenylalanyl
puromycin must come from
the phenylalanyl-tRNA
at the donor and the acceptor site, 248
cpm (495 X $. = 248) were assigned to each of these sites. The
phenylalanyl puromycin (5695 cpm) must come entirely from the
donor site. In the presence of the G factor, 6640 cpm of puromycin derivatives of [r%]phenylalanine
were formed (line 2 in
Table I). Of this, 89% (5910 cpm) and 11% (730 cpm) were
[4C]phenylalanyl
puromycin and diphenylalanyl
puromycin, respectively.
Thus, 215 cpm (5910 - 5695 = 215) of phenylalanyl
puromycin were formed due to the addition of G factor. We regard that this was bound at the acceptor site and translocated to
the donor site by the G factor. The diphenylalanyl
puromycin
formed due to the addition of G factor was 235 cpm (730 - 495 =
235). We assign 117 cpm (235 X + = 117) to eachsite.
The total
[r%]phenylalanyl-tRNA
bound to the donor site is therefore
5695 + 248 + 117 = 6060 cpm and that bound to the acceptor site
is 215 + 248 + 117 = 580 cpm. The sum of these values (6060 +
580 = 6640) represents total puromycin-reactive
[r%]phenplalanyl-tRNA
bound in the presence of 6 mM Mg++ (line 2 in Table
I).

and Puromycin

Issue of January

10, 1972
TABLE

Effect

S. Tanaka,

K. Igarashi,

and A. Kaji

III

01 acceptor
site aminoacyl-tRNA
on reaction
[14C]phenylalanyl-tRNA
with puromycin

of donor

site

Bound
[CphenylalanyltRNA

Aminoacyl-tRXA
for additional
binding at 13 nm Mg++

[~ClPhenylalanyl
puromycin derivatives formed
Without
G factor

G?%r

that binding

TABLE
IV
sites for N-acetylphenylalanyl-tRNA
those for phenylalanyl-tRNA

are same

as
The reaction mixture (0.1 ml) for the binding of [14C]aminoacyltRNA contained the following in micromoles: 8.0 Tris-HCl (pH
7.1), 4.0 KCl, various concentrations
of magnesium acetate, 30
rg of poly(U), 160 rg of ribosomes, and 16,800 cpm (38.2 rpmoles) of
[r4C]phenylalanyl-tRNA
or 16,800 cpm (38.2ppmoles) of N-acetyl[W]phenylalanyl-tRNA.
In some cases, a mixture of tRNA containing 38.2 ppmoles of [lzC]phenylalanyl-tRNAor
38.2 ppmoles of
N-acetyl[W]phenylalanyl-tRNA
was added. The reaction was
incubated for 25 min at 22 and the bound radioactive aminoacyltRNA was assayed by the Millipore
filter technique.
Bound aminoacyl-tRNA
Aminoacyl-tRNA

used

N-acetyl[W]phenylalanyltRNA
[14C]Phenylalanyl-tRNA.
N-Acetyl[14C]phenylalanyl-tRNA
and
[12C]phenylalanyl-tRNA.
.
[W]Phenylalanyl-tRNA
and [WIphenylalanyl-tRNA
[r4C]Phenylalanyl-tRNA
and N-acetyl[W]phenylalanyl-tRNA.

5rnM
MC+

13 nlY
Mg*

21 InM
1 Mg++

1102
1152

@m
4869
5802

6GG4
6612

498

2177

2381

503

3913

4290

1138

6043

6532

cm
No aminoacyl-tRNA..
[W]Phenylalanyl-tRNA..
N-Acetyl[W]phenylalanyl-tRNA.

736
644
634

483
107
111

541
468
136

phenylalanyl-tRNA
than with phenylalanyl-tRNA,
the rate of
formation of puromycin derivative was studied after a short
incubation period.
As can be seen from the figure, at the initial
period of the reaction a marked inhibition
of the puromycin
reaction of N-acetyl[C]phenylalanyl-tRNA
at the donor site was
observed. It should be pointed out that no G factor was added
in this experiment.
Thus, the inhibitory
effect of [r%]phenylalanyl-tRNA
at the acceptor site was stronger than that of Nacetyl[12C]phenylalanyl-tRNA
because the former inhibited the
puromycin reaction for two possible reasons, i.e. steric hindrance,
as well as formation of N-acetyldiphenylalanyl-tRNA,
while the
latter inhibited
only because of the possible steric hindrance.
These results are consistent with the concept that puromycin
attacks the donor site from the direction of the acceptor site.
The experiments described above were based on the assumption that the binding sites for N-acetylphenylalanyl-tRNA
were
the same as those for phenylalanyl-tRNA,
and N-acetylphenylalanyl-tRNA
behaved similarly
to phenylalanyl-tRNA
with
respect to the binding to ribosomes.
To ascertain that this
assumption was correct, the binding sites for N-acetylphenylalanyl-tRNA
were compared with those for phenylalanyl-tRNA.
As shown in Table IV, the amount of bound N-acetyl[W]phenylalanyl-tRNA
was approximately
equal to that of [r4C]phenylalanyl-tRNA
at three different Mg++ concentrations
tested.
The addition of an equal amount of [lZC]phenylalanyl-tRNA
reduced the binding of [14C]phenylalanyl-tRNA
as well as Nacetyl[14C]phenylalanyl-tRNA.
It is noted that in the presence
presence of high Mg++, [i2C]phenylalanyl-tRNA
inhibited more
efficiently the binding of N-acetyl[14C]phenylalanyl-tRNA
than
that of [14C]phenylalanyl-tRNA.
This suggests that the binding

affinity of phenylalanyl-tRNA
is higher than that of N-acetylphenylalanyl-tRNA
under these conditions.
When both [Clphenylalanyl-tRNA
and N-acetyl[14C]phenylalanyl-tRNA
were
present in the reaction mixture, the total amount of bound
radioactivity
was approximately
the same as that with [Clphenylalanyl-tRNA
or N-acetyl[14C]phenylalanyl-tRNA
alone.
These observations show that the binding site for N-acetylphenylalanyl-tRNA
is propably identical with that for phenylalanyl-tRNA
at low (5 to 6 mM) as well as high (13 mrvr) Mg+f
concentrations.
Thus, one can conclude that in the presence of
low Mg+f, N-acetylphenylalanyl-tRNA
binds mostly to the
donor site while it binds to both donor and acceptor sites at
high Mg+f.
DISCUSSION

Preceding reports on the action of protein synthesis inhibitor

(9) were based on the assumption that in the presence of low
Mg++, binding of phenylalanyl-tRNA
to ribosomes took place
mostly to the donor site while both donor and acceptor sites were
occupied with phenylalanyl-tRNA
in the presence of high Mg+f.
These assumptions were derived from the results of the NH2terminal analysis of the polyphenylalanine
formed from the
complex of [r4C]phenylalanyl-tRNA,
poly(U), and ribosomes at
different concentrations
of Mg++ (2). Although a similar conclusion was obtained from the studies on the puromycin reaction
of the ribosome-bound
phenylalanyl-tRNA
(7), it was necessary to
obtain further evidence for this notion by identifying the product
of puromycin reaction.
In this discussion, we designate the complex made in the presence of low Mg++ (5 to 6 mM) as complex A and that made in
the presence of high Mg++ (13 nlM) as complex B. The analysis
of the puromycin reaction product clearly established that the
bound phenylalanyl-tRNA
of complex A was mostly located at
the donor site because the addition of G factor resulted in very

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For the preparation

of the ribosomal complex having [WIphenylalanyl-tRNA
mostly at the donor site, the reaction mixture
(1 ml) contained 50 mM Tris-HCl (pH 7.2), 5 mM magnesium acetate, 28 mM NH&l, 12 mm KCI, 200pg of poly(U), 10.8 mg of ribosomes, and 9.7 X lo5 cpm (4.95 mg of tRNA) of [W]phenylalanyltRNA.
The mixture was incubated and the complex isolated as
described in the text. The reaction mixture (0.7 ml) for the additional binding of aminoacyl-tRNA
at 13 ml Mg++ contained 20
mM Tris-HCl (pH 7.2), 13 mM magnesium acetate, 40 mM NH&l
and, where indicated, 3 mg of mixture of tRNA containing 1.5
m@moles of [W]phenylalanyl-tRNA
or 2.3 mg of tRNA mixture
containing
1.2 mpmoles of N-acetyl[W]phenylalanyl-tRNA.
The mixture was incubated for 20 min at 22. For the puromycin
reaction of the complex thus prepared, 0.2 ml of the above mixture
was incubated for 60 min at 26 in 0.36 ml of a solution containing
40 mM Tris-HCl (pH 7.2), 170 mM NH&l; 13 mM magnesium acetate, 1 mM puromycin, 0.2 mM GTP, and where indicated 13 rg of
G factor. The puromycin derivative formed was counted as described in the text.

Evidence

49

50

Action of Tetracycline and Puromycin

247, No. 1

rate, these observations indicate that puromycin attacks the

donor site peptidyl-tRNA
from the side of the acceptor site. Although there is no direct evidence that puromycin is actually at
the acceptor site when it reacts with the donor site peptidyltRNA, our present observation is consistent with the current
notion that puromycin will react with peptidyl transferase in a
way similar to the reaction of acceptor site aminoacyl-tRNA.
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A. (1967) Proc. Nut. Acad. Sci.

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HEINTZ,
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SARKER, S., AND THACH,

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small, if any, stimulation

of the formation of diphenylalanyl
On the other hand, complex B yielded diphenylpuromycin.
alanyl puromycin
as well as phenylalanyl
puromycin
in the
presence of G factor.
These results give further support of the
previous notion (2) that in the presence of low MgH (condition
A) only the donor sites are occupied with phenylalanyl-tRNA,
whereas both acceptor and donor sites ore occupied with phenylalanyl-tRNA
in the presence of high Mg+f (condition B).
With the analysis of the puromycin derivatives formed from
the bound phenylalanyl-tRNA,
it became clear that, in confumation of our previous results with NHz-terminal
analysis of
polyphenylalanine
(7), tetracycline
inhibited
significantly
the
bindmg of phenylalanyl-tRNA
at the donor site as well as the
acceptor site in the presence of high Mg*
(condition B). In the
presence of low Mg* and T factor the action of tetracycline was
relatively specific to the acceptor site. It should be pointed out,
however, that in the normal situation, the donor site is occupied
with either unesterified
tRNA or peptidyl-tRNA;
thus the
possiblity that the aminoacyl-tRNA
directly binds to the donor
site does not exist in the normal protein synthesis.
Only when
the empty ribosomes are presented to an aminoacyl-tRNA
with
synthetic messenger RNA, such a situation takes place.
The presence of N-acetylphenylalanyl-tRNA
or phenylalanyltRNA at the acceptor site has a strong inhibitory effect on the
puromycin reaction of the donor site-bound [14C]phenylalanyltRNA.
The inhibition
by the presence of [WlphenylalanyltRNA at the acceptor site is due to two reasons. The first is
formation
of [14C]phenylalanyl-[W]phenylalanyl-tRNA
bound
to the acceptor site. In support of this, a marked increase of
the puromycin reaction was observed by the addition of G factor.
The second reason for the inhibition is a possible steric hindrance
caused by the presence of aminoacyl-tRNA
at the acceptor site.
This notion was supported by the observation that the presence
of N-acetyl[W]phenylalanyl-tRNA
at the acceptor site had
strong inhibitory effect on the puromycin reaction of the donor
site-bound [14C]phenylalanyl-tRNA.
The inhibitory effect of the
acceptor site-bound N-acetyl[12C]phenylalanyl-tRNA
could be
due to its effect on the conformation of the ribosome.
At any

Vol.